X-Linked Macrocytic Dyserythropoietic Anemia in Females with an ALAS2 Mutation

Amendment history: Corrigendum (January 2020) X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation

Vijay G. Sankaran, … , David F. Bishop, David P. Steensma

J Clin Invest. 2015;125(4):1665-1669. https://doi.org/10.1172/JCI78619.

Brief Report Hematology

Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5′- aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5′-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell- nonautonomous effects. Moreover, our findings highlight the value of whole-exome […]

Find the latest version: https://jci.me/78619/pdf The Journal of Clinical Investigation Brief Report

X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation

Vijay G. Sankaran,1,2 Jacob C. Ulirsch,1,2 Vassili Tchaikovskii,3 Leif S. Ludwig,1,2,4,5 Aoi Wakabayashi,1,2 Senkottuvelan Kadirvel,3 R. Coleman Lindsley,6 Rafael Bejar,6 Jiahai Shi,7 Scott B. Lovitch,8 David F. Bishop,3 and David P. Steensma6 1Division of Hematology/Oncology, Boston Children’s Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. 2Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. 3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, Mount Sinai Medical Center, New York, New York, USA. 4Institute for Chemistry and Biochemistry, Freie Universität, Berlin, Germany. 5Charité-Universitätsmedizin, Berlin, Germany. 6Division of Hematologic Malignancies, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, USA. 7Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. 8Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA.

Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5′-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5′-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell- nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease.

Introduction Results and Discussion Macrocytic anemia and abnormal dysplastic erythropoiesis The proband (family member II-2) (Figure 1E) was a 32-year-old (herein referred to as dyserythropoiesis) are features found in a woman with a history of atrial septal defect who had been noted to number of congenital and acquired conditions, including congen- have an anemia in childhood of unclear etiology. She had come to ital dyserythropoietic anemias (CDAs), specific mitochondrial clinical attention 7 years earlier, when a routine blood count during disorders, myelodysplastic syndromes (MDS), and megaloblastic her first pregnancy revealed an anemia with a hematocrit of 24% to anemias (1, 2). While numerous etiologies are known for this class 26% and a mean corpuscular volume of 114 to 122 femtoliters (Sup- of disorders, some cases lack a clear explanation (3). An improved plemental Table 1; supplemental material available online with this understanding of the etiology of these disorders will provide article; doi:10.1172/JCI78619DS1) that was unresponsive to iron and additional insight into the mechanisms of abnormal blood cell vitamin B12 supplementation. Delivery occurred at full term and production in humans (4). was complicated by placenta accreta with concomitant blood loss, We hypothesized that certain cases of this type of anemia and the proband required an rbc transfusion for the first time. Three either represent new genetic conditions or, alternatively, are due years later, the proband delivered healthy twins and experienced a to the phenotypic variation in genes previously associated with massive hemorrhage 2 weeks after the delivery, resulting in a drop of anemia. We identified a female proband with a macrocytic dys- her hematocrit to 17% that required an additional transfusion. erythropoietic anemia since childhood and evidence of iron over- At that time, it was noted that the proband had a macro- load, who had undergone an extensive, but unrevealing, clinical cytic anemia and an elevated serum ferritin level (800 ng/ml), evaluation. The proband’s sister and mother were also noted to prompting referral to a hematologist. A BM biopsy revealed trilin- have macrocytic anemia, supporting a potential genetic etiology. eage hematopoiesis with dyserythropoiesis (Figure 1, A–D). The We therefore performed whole-exome sequencing and func- proband had a normal BM karyotype and MDS/acute myeloid tional studies to identify and understand the basis of the anemia leukemia FISH panel (evaluating chromosomes 5, 7, 8, 13, 20, and observed in this family. X). According to the report, Prussian blue reaction on the initial BM aspirate showed no excess iron or sideroblasts. A liver biopsy revealed an iron index of 11 (normal, <1), and HFE genetic testing Conflict of interest: The authors have delcared that no conflict of interest exists. Submitted: August 26, 2014; Accepted: January 8, 2015. revealed no mutations. Therapeutic phlebotomy was attempted, Reference information: J Clin Invest. 2015;125(4):1665–1669. doi:10.1172/JCI78619. but was limited by symptomatic anemia.

jci.org Volume 125 Number 4 April 2015 1665 Brief REport The Journal of Clinical Investigation

Figure 1. Identification of an ALAS2 mutation in a family with macrocytic anemia and dyserythropoiesis. (A) BM aspirate from the proband (II-2) with erythroid hyperplasia. (B) Prussian blue staining revealed rare erythroblasts with siderotic granules. (C) BM aspirate with dyserythropoiesis. (D) Core biopsy demonstrating erythroid hyperplasia and dyserythropoiesis. (E) Pedigree of the family, with affected individuals highlighted by half-filled circles. Arrows highlight those individuals who underwent whole-exome sequencing. (F) Sanger sequencing traces around X chromosome position 55042086 (hg19 coordinates) in 2 affected individuals. Original magnification, ×100 A( and B) and ×2000 (C and D).

ure 1E). T2* MRI was performed on the proband and revealed an estimated liver iron concentration of greater than 350 μmol/g (normal, <35 μmol/g). In addition, the proband had a horseshoe kidney and a mildly enlarged spleen, measuring 14.8 cm. A trial with the iron chelator deferasirox was attempted but was not tolerated, and therefore another trial of phlebotomy with a target hemoglo- bin level of 10 g/dl was initiated. Since the clinical features of this patient suggested the possibility of CDA, targeted sequencing of CDAN1, SEC23B, and KLF1 was performed, but no mutations were identified (5). The proband and family members then consented to participate in a whole-exome sequencing study (6, 7). We used an automated pipeline to identify novel coding and the loss-of-function (LOF) variants found in the 3 affected individuals from this family (I-1, II-2, and II-4), but not in the unaffected Laboratory testing of the proband, her sister (family member daughter (III-1) (6). Each individual had a total of approximately II-4), and mother (family member I-2) demonstrated rbc mac- 200 novel coding and LOF variants (Supplemental Table 2). A sin- rocytosis (Figure 1E and Supplemental Table 1). The brother and gle notable variant fit the model of complete penetrance and was daughters of the proband had normal complete blood counts (Fig- found to be a coding mutation in the X chromosomal gene ALAS2.

Figure 2. Severe LOF with the ALAS2 Y365C mutation and lack of highly skewed X inactivation in female mutation carriers. (A) Model of ALAS2 shows PLP highlighted in blue and the Y or C amino acid at position 365 highlighted in red. (B) SDS-PAGE gel of WT and mutant ALAS2. Lanes 1 and 8 contain the protein standards, while lanes 2–4 and 5–7 contain WT and mutant ALAS2 protein samples, respectively. Lanes 2 and 5 show partially purified samples after amylose affinity chromatography, lanes 3 and 6 show results after factor Xa digestion, and lanes 4 and 7 show results after gel filtration chromatography. Thin vertical lines in this composite figure separate noncontiguous lanes in the 2 original gels. C( ) Chromatographic profiles for purification of WT and mutant ALAS2 by size exclusion (absorbance at 280 nm is shown in milliabsorbance units [mAU]). (D) Quantification of HUMARA results in all affected individuals showing WT and mutant X chromosomes. (E) Sanger sequencing traces of genomic DNA (gDNA) and cDNA derived from reticulocyte mRNA from the proband (II-2) for ALAS2, with an arrow highlighting the mutation.

1666 jci.org Volume 125 Number 4 April 2015 The Journal of Clinical Investigation Brief Report

mutant purified to homogeneity had a 70-fold lower affinity for Table 1. Properties of WT and mutant ALAS2 PLP than did the WT enzyme (Table 1). The ALAS2 C365 mutant Property WT (Y365) C365 mutant had enzyme kinetics similar to that of the WT enzyme (Table 1). Yield (%) 17.7 ± 1.3 (n = 4) 5.5 ± 1.3 (n = 2) However, the mutant enzyme was unstable, consistent with the Specific activity (U/mg) 90,700 62,100 notion that PLP binding plays a key role in stabilizing ALAS2 (17,

Vmax SCoA (U/mg) 96,200 78,800 18). When the enzymes were expressed as maltose-binding protein

Vmax Gly (U/mg) 85,000 73,700 (MBP) fusions, the yield of the mutant enzyme in the initial crude

Hilln SCoA 1.4 1.3 extract was only 3.3% of the WT enzyme yield (Figure 2B). After

Km SCoA (μM) 60 33 removal of the MBP and purification by liquid chromatography

Km Gly (mM) 8.0 8.4 (Figure 2C), only 5.5% of the mutant enzyme activity was recov- K PLP (nM) 11 737 m ered compared with 17.9% of the WT enzyme activity (Table 1). t in 10 uM PLP (min) 7.57 ± 0.96 1.24 ± 0.15 1/2 Overall, the yield relative to that of WT was only 0.2% — lower than t in 100 uM PLP (min) 15.6 ± 1.3 1.46 ± 0.15 1/2 the residual activities in classic cases of XLSA (15). Furthermore, Specific activities were for a single purification experiment, the kinetic the mutant enzyme was 6-fold less stable than the WT enzyme at values of V , Hill , and K were derived from Lineweaver-Burk plots max n m 50°C (Table 1). Under conditions of increased PLP, the mutant was of single kinetic experiments, and the half-life data at 50°C were the 11-fold less stable because the PLP stabilized the WT, but not the average and SD of 3 independent experiments for each enzyme at each mutant, enzyme (Table 1). Together, these findings indicate that PLP concentration. Gly, glycine; Hill , derived Hill coefficient; K , substrate n m the Y365C mutation markedly impairs PLP binding, which may concentration; SCoA, succinyl CoA; V , maximum reaction velocity. max account for some or all of the substantially reduced stability of the ALAS2 enzyme. Severe LOF due to this mutation may explain why no male carriers were identified in the family (Figure 1E) and why This A-to-G variant was found at position 55042086 on the X chro- the proband’s anemia did not improve with pyridoxine therapy. mosome (hg19 coordinates), resulting in a coding change of Y365C As only female carriers were found to be affected, we reasoned in the ALAS2 protein (Figure 1F and Supplemental Figure 1). that there may have been highly skewed X inactivation present in The enzyme encoded by the ALAS2 gene plays a critical role the individuals in this family. We were surprised to find that 62%– in heme biosynthesis, and mutations in this gene are known to 82% of the active X chromosomes in the proband and other affected cause a microcytic sideroblastic anemia in male carriers and in family members contained the ALAS2 mutant, as assessed by the females with skewed X inactivation (X-linked sideroblastic ane- human androgen receptor gene polymorphism assay (HUMARA) mia [XLSA]; Online Mendelian Inheritance in Man [OMIM] entry for X inactivation in whole-blood genomic DNA (Figure 2D). no. 300571) (8–15). However, mutations in this gene have only These values were all within 2 SD of the mean X inactivation ratios rarely been associated with macrocytic anemia or dyserythropoiesis (14). We confirmed that this mutation was found in all affected individuals through Sanger sequencing (Figure 1F), while the unaffected individuals in this family lacked this mutation, further supporting its causality for the phenotype. Additionally, another BM aspirate from the proband revealed occasional cells with siderotic granules, while nearly all erythroid cells showed dyserythropoietic features, even in the absence of such granules (Figure 1, A–D). By modeling this novel ALAS2 Y365C mutation in the structure of the Rhodobacter capsulatus homolog, we noted that Y365 fits within a hydrophobic core critical for binding the essential cofactor pyridoxal 5′-phosphate (PLP) (Figure 2A and ref. 16). The C365 mutation would disrupt this hydrophobic core and would therefore be predicted to reduce the ability of ALAS2 to bind to PLP (Fig- ure 2A). Consistent with this idea, we found that the ALAS2 C365

Figure 3. X-linked dominant LOF ALAS2 mutations can result in macrocytosis and dyserythropoiesis through cell-nonautonomous effects. (A) Sequencing of cDNA derived from cultured erythroid cells from the proband showed skewing toward the WT ALAS2 allele at the late stages of erythroid differentiation. Cells began as progenitors (day 5), became intermediate erythroblasts (day 9), and transitioned to orthochromatic erythroblasts (day 14). By late erythropoiesis, only a trace amount of the mutant allele was detectable. (B) Model showing how developing erythroid progenitors and precursors compete for space within the BM. The active X chromosome expressed in each group of cells (brown and pink for the mutant and WT, respectively) is shown in blue, with some cells expressing the mutant (red) and others expressing the WT (green) allele.

jci.org Volume 125 Number 4 April 2015 1667 Brief REport The Journal of Clinical Investigation observed in healthy adult females (19). We obtained independent Our findings illustrate how cell-nonautonomous defects could confirmation for a lack of highly skewed X inactivation by digesting arise as a result of a mutant ALAS2 allele on one X chromosome, genomic DNA from the affected family members with the methy- even in the absence of significantly skewed X inactivation. Few lation-sensitive restriction enzyme HpaII and noted no change in examples exist of such mutations in human disease (23). We the intensity of the ALAS2 mutation by Sanger sequencing (Fig- have only identified a single family with this phenotype, but as ure 1F and Supplemental Figure 2). The proband, who was the more cases of macrocytic dyserythropoietic anemia are studied, most severely affected, had the greatest extent of skewing toward it is likely that other similar mutations in ALAS2 will be identi- the mutant allele, at 82% (Figure 2D). Collectively, these findings fied. While XLSA is typically a dimorphic, microcytic anemia suggest that the presence of some erythroid cells expressing WT in females, sufficiently deleterious ALAS2 mutations can result ALAS2 in the patients’ BM was insufficient to compensate for the in macrocytic anemia without a dimorphic population in such presence of erythroid cells expressing the ALAS2 mutation. females. We illustrate how defining such mutations using modern While most ALAS2 mutations causing sideroblastic anemia genomic sequencing can help lead to further insight into human are partial LOF alleles affecting heme biosynthesis (12, 13, 15), disease and hematopoiesis (7). complete ALAS2 LOF in mouse models results in an early block of erythropoiesis at approximately the proerythroblast or basophilic Methods erythroblast stages (20). Consistent with these results, when we Further information can be found in the Supplemental Methods. sequenced peripheral blood reticulocyte mRNA samples, we only The whole-exome sequencing data on the family members are detected the presence of transcripts with the WT allele, while the available in the dbGaP database (http://www.ncbi.nlm.nih.gov/gap) mutant allele was readily detectable in genomic DNA (Figure under the accession number phs000474.v2.p1. 2E). To confirm these findings, peripheral blood mononuclear Statistics. All pairwise comparisons were performed using the cells from the proband were cultured in medium that promotes 2-tailed Student’s t test, unless otherwise indicated. Differences were erythroid differentiation. We found that as the cells transitioned considered significant if the P value was less than 0.05. from the early progenitor stages to terminally differentiated ery- Study approval. All family members provided written informed throblasts, there was selection against cells expressing the mutant consent to participate in this study. The IRBs of the Dana-Farber Can- ALAS2 mRNA, leaving only cells expressing mRNA from the WT cer Institute, Boston Children’s Hospital, and Massachusetts Institute allele (Figure 3A). Thus, the X inactivation and mRNA sequencing of Technology approved the study protocols. studies show that while there is initially a mixed population of erythroid progenitors and early precursors that express both ALAS2 Acknowledgments alleles, as cells undergo erythroid differentiation, there is a loss of We are grateful to the family for their interest in this study. We nonviable cells expressing the mutant allele, with a survival advan- thank D. Nathan, S. Lux, C. Brugnara, F. Briccetti, M. Wester- tage for cells expressing the WT allele (Figure 3B and ref. 14). This man, J. Hirschhorn, G. Evrony, R. Do, D. Bennett, and M. Burns pressure to produce a normal output of erythroid cells may affect for their valuable advice and support. This work was supported differentiation and result in skipped cell divisions to ensure ade- by NIH grants R21 HL120791 and R01 DK103794 (to V.G. San- quate erythroid output, which would explain the macrocytosis karan) and by a grant from the Edward P. Evans Foundation (to and dyserythropoiesis observed among those cells expressing WT D.P. Steensma). ALAS2 alleles (ref. 21 and Figure 3B). It is unclear whether there is a connection between the ALAS2 Address correspondence to: Vijay G. Sankaran, Boston Chil- mutation and the proband’s congenital defects in other tissues dren’s Hospital, 3 Blackfan Circle, CLS 03001, Boston, Massa- (i.e., atrial septal defect, horseshoe kidney) and her abnormal pla- chusetts 02115, USA. Phone: 617.919.6270; E-mail: sankaran@ centation, which were not present in other family members. We broadinstitute.org. Or to: David P. Steensma, Dana-Farber Can- examined known genetic causes of iron overload in the proband, cer Institute, 450 Brookline Avenue, Boston, Massachusetts including mutations in the ALAS2, HAMP, HFE, HFE2, SLC40A1, 02215, USA. Phone: 617.632.3712; E-mail: David_Steensma@ and TFR2 genes, and only the mutation in ALAS2 was identified. dfci.harvard.edu. We also observed low levels of hepcidin in the blood of the proband (Supplemental Table 1), consistent with the expected reduction of Rafael Bejar’s present address is: University of California, San hepcidin that occurs in the context of ineffective erythropoiesis (22). Diego, La Jolla, California 92093, USA.

1. Hoffbrand V, Provan D. ABC of clinical Dev. 2013;23(3):339–344. 8. Cotter PD, Baumann M, Bishop DF. Enzymatic haematology. Macrocytic anaemias. BMJ. 5. Iolascon A, Heimpel H, Wahlin A, Tamary H. defect in “X-linked” sideroblastic anemia: 1997;314(7078):430–433. Congenital dyserythropoietic anemias: molec- molecular evidence for erythroid delta-aminole- 2. Davenport J. Macrocytic anemia. Am Fam Physi- ular insights and diagnostic approach. Blood. vulinate synthase deficiency. Proc Natl Acad Sci cian. 1996;53(1):155–162. 2013;122(13):2162–2166. U S A. 1992;89(9):4028–4032. 3. Younes M, Dagher GA, Dulanto JV, Njeim M, 6. Sankaran VG, et al. Exome sequencing identifies 9. Bottomley SS, May BK, Cox TC, Cotter PD, Kuriakose P. Unexplained macrocytosis. South GATA1 mutations resulting in Diamond-Blackfan Bishop DF. Molecular defects of erythroid 5-ami- Med J. 2013;106(2):121–125. anemia. J Clin Invest. 2012;122(7):2439–2443. nolevulinate synthase in X-linked sideroblastic 4. Sankaran VG, Orkin SH. Genome-wide associa- 7. Sankaran VG, Gallagher PG. Applications of anemia. J Bioenerg Biomembr. 1995;27(2):161–168. tion studies of hematologic phenotypes: a win- high-throughput DNA sequencing to benign 10. Cotter PD, et al. Four new mutations in the dow into human hematopoiesis. Curr Opin Genet hematology. Blood. 2013;122(22):3575–3582. erythroid-specific 5-aminolevulinate synthase

1668 jci.org Volume 125 Number 4 April 2015 The Journal of Clinical Investigation Brief Report

(ALAS2) gene causing X-linked sideroblastic ane- unfortunate skewed X-chromosome inactivation original family described by Cooley. Blood. mia: increased pyridoxine responsiveness after patterns. Blood Cells Mol Dis. 2006;37(1):40–45. 1994;84(11):3915–3924. removal of iron overload by phlebotomy and 15. Ducamp S, et al. Sideroblastic anemia: molecu- 19. Amos-Landgraf JM, et al. X chromosome-in- coinheritance of hereditary hemochromatosis. lar analysis of the ALAS2 gene in a series of 29 activation patterns of 1,005 phenotypi- Blood. 1999;93(5):1757–1769. probands and functional studies of 10 missense cally unaffected females. Am J Hum Genet. 11. Cazzola M, May A, Bergamaschi G, Cerani P, mutations. Hum Mutat. 2011;32(6):590–597. 2006;79(3):493–499. Rosti V, Bishop DF. Familial-skewed X-chromo- 16. Astner I, Schulze JO, van den Heuvel J, Jahn D, 20. Harigae H, et al. Aberrant iron accumulation and some inactivation as a predisposing factor for Schubert WD, Heinz DW. Crystal structure of oxidized status of erythroid-specific Δ-aminole- late-onset X-linked sideroblastic anemia in car- 5-aminolevulinate synthase, the first enzyme vulinate synthase (ALAS2)-deficient definitive rier females. Blood. 2000;96(13):4363–4365. of heme biosynthesis, and its link to XLSA in erythroblasts. Blood. 2003;101(3):1188–1193. 12. Bergmann AK, et al. Systematic molecular humans. EMBO J. 2005;24(18):3166–3177. 21. Sankaran VG, et al. Cyclin D3 coordinates genetic analysis of congenital sideroblastic 17. Bishop DF, Tchaikovskii V, Hoffbrand AV, Fraser the cell cycle during differentiation to regu- anemia: evidence for genetic heterogeneity and ME, Margolis S. X-linked sideroblastic anemia late erythrocyte size and number. Genes Dev. identification of novel mutations. Pediatr Blood due to carboxyl-terminal ALAS2 mutations that 2012;26(18):2075–2087. Cancer. 2010;54(2):273–278. cause loss of binding to the β-subunit of suc- 22. Kautz L, Jung G, Valore EV, Rivella S, Nemeth 13. Fleming MD. Congenital sideroblastic anemias: cinyl-CoA synthetase (SUCLA2). J Biol Chem. E, Ganz T. Identification of erythroferrone as iron and heme lost in mitochondrial translation. 2012;287(34):28943–28955. an erythroid regulator of iron metabolism. Nat Hematology Am Soc Hematol Educ Program. 18. Cotter PD, Rucknagel DL, Bishop DF. X-linked Genet. 2014;46(7):678–684. 2011;2011:525–531. sideroblastic anemia: identification of the 23. Migeon BR. The role of X inactivation and cellu- 14. Aivado M, et al. X-linked sideroblastic anemia mutation in the erythroid-specific Δ-ami- lar mosaicism in women’s health and sex-specific associated with a novel ALAS2 mutation and nolevulinate synthase gene (ALAS2) in the diseases. JAMA. 2006;295(12):1428–1433.

jci.org Volume 125 Number 4 April 2015 1669